The present invention relates to compositions comprising paroxonase 3 genes and polypeptides, in particular to compositions comprising rabbit PON3 genes and polypeptides. The present invention also provides methods for using PON3 genes and peptides in the treatment of endotoxemia, oxidative damage, chemical toxicity, and other conditions.
Endotoxic shock is a condition, often fatal, provoked by the release of lipopolysaccharide (LPS) from the outer membrane of most gram negative bacteria (e.g., Escherichia coli; Salmonella tymphimurium). One example of a condition involving Endotoxic shock is sepsis. Sepsis is a major cause of morbidity and mortality in humans and other animals. It is estimated that 400,000-500,000 episodes of sepsis resulted in 100,000-175,000 human deaths in the U.S. alone in 1991. Sepsis has become the leading cause of death in intensive care units among patients with non-traumatic illnesses (Machiedo et al., Surg. Gyn. and Obstet., 152:757-759 [1981]). It is also the leading cause of death in young livestock, affecting 7.5-29% of neonatal calves (Morris et al., Am. J. Vet. Res., 47:2554-2565 [1986]), and is a common medical problem in neonatal foals (Hoffman et al., J. Vet. Int. Med., 6:89-95 [1992]). Despite the major advances of the past several decades in the treatment of serious infections, the incidence and mortality due to sepsis continues to rise (Wolff, New Eng. J. Med., 324:486-488 [1991]).
Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction. Sepsis can result from septicemia (i.e., organisms, their metabolic end-products or toxins in the blood stream), including bacteremia (i.e., bacteria in the blood), as well as toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxin in the blood). The term xe2x80x9cbacteremiaxe2x80x9d includes occult bacteremia observed in young febrile children with no apparent foci of infection. The term xe2x80x9csepsisxe2x80x9d also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitemia (i.e., helminthic or protozoan parasites in the blood). Thus, septicemia and septic shock (acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate) may be caused by a number of organisms.
The systemic invasion of microorganisms presents two distinct problems. First, the growth of the microorganisms can directly damage tissues, organs, and vascular function. Second, toxic components of the microorganisms can lead to rapid systemic inflammatory responses that can quickly damage vital organs and lead to circulatory collapse (i.e., septic shock) and oftentimes, death.
There are three major types of sepsis characterized by the type of infecting organism. Gram-negative sepsis is the most common and has a case fatality rate of about 35%. The majority of these infections are caused by Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such as the staphylococci and streptococci are the second major cause of sepsis. The third major group includes the fungi, with fungal infections causing a relatively small percentage of sepsis cases, but with a high mortality rate.
Many of these infections are acquired in a hospital setting and can result from certain types of surgery (e.g., abdominal procedures), immune suppression due to cancer or transplantation therapy, immune deficiency diseases, and exposure through intravenous catheters. Sepsis is also commonly caused by trauma, difficult newborn deliveries, and intestinal torsion (especially in dogs and horses).
Many patients with septicemia or suspected septicemia exhibit a rapid decline over a 24-48 hour period. Thus, rapid methods of diagnosis and treatment delivery are essential for effective patient care. Unfortunately, a confirmed diagnosis as to the type of infection traditionally requires microbiological analysis involving inoculation of blood cultures, incubation for 18-24 hours, plating the causative organism on solid media, another incubation period, and final identification 1-2 days later. Therefore, therapy must be initiated without any knowledge of the type and species of the pathogen, and with no means of knowing the extent of the infection.
It is widely believed that anti-endotoxin antibody treatment administered after sepsis is established may yield little benefit because these antibodies cannot reverse the inflammatory cascade initiated by endotoxin. In addition, the high cost of each antibody (Centoxin HA-1A was expected to cost $3700 per 100 mg dose) would limit physicians"" use of a product where no clear benefit has been demonstrated (K. A. Schulman et al., JAMA, 266:3466-3471 [1991]). Of course, these endotoxin antibodies only target gram-negative sepsis; no equivalent antibodies exist for the array of gram-positive organisms and fungi.
With new knowledge regarding the effects of endotoxin on host inflammatory responses, other therapies are being attempted. For example, an IL-1 receptor antagonist has been identified that occupies the same receptor site as IL-1, but mediates no biological effect. Blockage of the IL-1 receptor with this molecule can reduce mortality from endotoxin shock. (Ohlsson et al., Nature, 348:550-552 [1990]). While the IL-1 receptor antagonist appears to be well-tolerated, the required dosage is extremely large (over 100 mg of recombinant protein per kg of body weight is infused over a period of hours to days). For human therapy, the 8-10 grams of recombinant protein anticipated to be required is likely to be extremely costly (several thousand dollars).
Clearly, there is a great need for agents capable of preventing and treating sepsis. It would be desirable if such agents could be administered in a cost-effective fashion. Furthermore, approaches are needed to combat all forms of sepsis, not just gram-negative cases.
The present invention relates to compositions comprising paroxonase 3 genes and polypeptides, in particular to compositions comprising rabbit PON3 genes and polypeptides. The present invention also provides methods for using PON3 genes and peptides in the treatment of endotoxemia, oxidative damage, chemical toxicity, and other conditions.
For example, the present invention provides compositions comprising a nucleic acid sequence comprising at least a portion of a gene encoding rabbit PON-3 (e.g., a portion encoding a biologically active PON-3 polypeptide). In some embodiments, the nucleic acid sequence comprises SEQ ID NO:7 or a portion of SEQ ID NO:7 (e.g., a portion encoding a biologically active PON-3 polypeptide such as the PON-3 functional domains described herein). In certain embodiments, the nucleic acid sequence comprises a truncation of SEQ ID NO:7. In some embodiments, the nucleic acid comprises a PON-3 variant sequence. Although the present invention is not limited by the identity of the variant sequences, in some embodiments the variants include, but are not limit to, SEQ ID NOs:9, 11, 13, 15, and 17 or peptide sequences 10, 12, 14, 16, and 18.
The present invention also provides polypeptides encoded by the above nucleic acid sequences. For example, the present invention provides the polypeptide SEQ ID NO:8. In preferred embodiments of the present invention, the present invention provides biologically active rabbit PON3 polypeptides or polypeptide fragments. In certain embodiments, the polypeptides further comprise non-rabbit PON-3 polypeptide sequences (e.g., a biologically active rabbit PON3 polypeptide is provided as a chimera with a human PON3 polypeptide sequence).
The present invention also provides methods comprising providing: a biologically active PON3 polypeptide or polypeptide fragment (e.g., including, but not limited to any of the above peptides), a host, and a delivery system; and administering the biologically active rabbit PON3 polypeptide or fragment to the host using the delivery system. The present invention is not limited by the nature of the host. For example, host include, but are not limited to humans and non-human mammals. The host may be treated for any number of reasons. In some embodiments, the host is further treated with other PON polypeptides (e.g., PON-1 and/or PON-2 polypeptides), for example, in a mixture with PON-3. While the present invention is not limited by the nature of the host, in preferred embodiments, the host is a host suspected of having sepsis or known to have sepsis and hosts suspected as being susceptible to sepsis.